Department of Microbiology and Molecular Genetics, School of Medicine, University of California Irvine, B240 Medical Sciences I, Irvine, CA 92697, USA.
J Mol Biol. 2010 Jun 25;399(5):696-706. doi: 10.1016/j.jmb.2010.04.035. Epub 2010 Apr 24.
3'-Terminal uridylyl transferases (TUTases) selectively bind uridine 5'-triphosphate (UTP) and catalyze the addition of uridine 5'-monophosphate to the 3'-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.
3′-末端尿苷酰转移酶（TUTases）选择性结合尿苷 5′-三磷酸（UTP），并在模板非依赖性方式下催化尿苷 5′-单磷酸添加到 RNA 底物的 3′-羟基。RNA 编辑 TUTase 1 和 RNA 编辑 TUTase 2（RET2）在尿苷插入/缺失 RNA 编辑中发挥核心作用，这是原生动物线粒体 RNA 加工的重要组成部分。尽管所有已知的 TUTases 都很容易区分保守的 N 端（催化）结构域和 C 端（核苷酸碱基识别）结构域，但核苷酸特异性、RNA 底物偏好性、连续性、四级结构和辅助结构域在具有不同生物学功能的酶之间差异显著。RET2 作为 RNA 编辑核心复合物的亚基，执行指导 RNA 依赖性的线粒体 mRNA 中的 U 插入。通过将 RET2 活性的突变效应与重组蛋白和 RNA 编辑核心复合物亚基相关联，并与基于 RNAi 的基因敲入表型进行关联，我们评估了 RET2 中的 UTP 和 RNA 结合位点。在这里，我们证明了关键 UTP 结合和金属离子配位残基的功能保守性，并确定了参与 RNA 底物识别的氨基酸。位于 UTP 结合位点附近的保守精氨酸残基 144 和 435 对于 RET2 在单链和双链 RNA 上的活性以及体内功能至关重要。对类似于指导 RNA/mRNA 双链的双链 RNA 的识别进一步通过 RET2 特异性中间结构域上的多点接触得到促进。
